(C) Overexpression of PPT1 in both SH-p
(C) Overexpression of PPT1 in both SH-p.wtCLN1 cells and cell lines harboring a missense mutation was confirmed by immunoblotting analysis. were predicted to be hampered in the wtoverexpressing cell line, and these findings were corroborated by morphological investigations. Palmitoylation survey identified 113 SL910102 palmitoylated protein-encoding genes in SH-p.wtCLN1, including 25 ones simultaneously assigned to axonal growth and synaptic compartments. A remarkable decrease in the expression of palmitoylated proteins, functionally related to axonal elongation (GAP43, CRMP1 and NEFM) and of the synaptic marker SNAP25, specifically in SH-p.wtCLN1 cells was confirmed by immunoblotting. Subsequent, bioinformatic network survey of DEGs assigned to the synaptic annotations linked 81 DEGs, including 23 ones encoding for palmitoylated proteins. Results obtained in this experimental setting outlined two affected functional modules (connected to the axonal and synaptic compartments), which can be associated with an altered gene dosage of wtknockout mice and patients with CLN1 disease. models (including zebrafish and mice) and systems have been extensively used to dissect out the role of PPT1 in neuronal tissues (Lyly et al., 2007; Bond et al., 2013; Faller et al., 2015). Among cellular models, neuroblastoma cells have been acknowledged as a useful system to investigate the effects of NCL genes expression on neuronal functions and protein interactions. Overexpression of protects LAN-5 neuroblastoma cells from ceramide-induced apoptosis (Cho and Dawson, 2000), whereas antisense treatment (leading to a reduced expression of PPT1) increased the susceptibility to undergo cell death (Cho et al., 2000a). Recently, SH-SY5Y neuroblastoma cells overexpressing either or have been also used to recognize putative interacting proteins of PPT1 and CLN3/CLN5 crosstalk by proteomics approach (Scifo et al., 2013, 2015a,b). The aim of this study was to recognize SL910102 molecular signatures and functional modules connected with overexpressed in human neuronal SL910102 cellular system. We utilized SH-SY5Y neuroblastoma cells (differentiated into a neuronal-like phenotype, Pezzini et al., 2017) to overexpress wtand a selection of disease related mutations previously detected in CLN1-affected children. The differentiated cell lines underwent whole transcriptomic profiling by RNA-seq, to identify differentially expressed genes (DEGs) which are functionally related to the overexpression of wild-type or mutated PPT1. Following bioinformatic investigations, we focused on DEGs involved in palmitoylation of neuronal proteins as well as related cellular functions. Interestingly, genes coding for palmitoylated proteins assigned to neuronal functions, such as axonal growth, and to the synaptic compartment were the most significantly expressed. Moreover, to identify potential therapeutic targets for CLN1 disease, we aimed to demonstrate possible links with other NCL genes, particularly and (Haltia and Goebel, 2013). Materials and Methods Cell Culture Human neuroblastoma SH-SY5Y cells (catalog number #94030304 European Collection of Cell Cultures) were cultured in DMEM-High glucose medium supplemented with 15% Foetal Bovine Serum (FBS), 2 mM L-glutamine and 1% non-essential amino acids (all from Euroclone), at 37C in humified atmosphere with 5% CO2, as described (Pezzini et al., 2017). Production of PPT1 Constructs, Cell Transfection and Generation of Stable Cell SL910102 Lines We focused our attention on mutations described in Mediterranean patients affected by classical and variant CLN1 disease. Specifically, we selected three different missense mutations (c.665T C/p.L222P, c.541G A/p.V181M and c.541G T/p.V181L), a deletion of the second exon (c.125_235del/p.G42_E78del) in HSPC150 which the ORF is maintained and a one-base insertion (c.169dupA/p.M57Nfs*45) predicting a frameshift, and a premature stop codon at residue 101 (Santorelli et al., 1998). Wild-type and mutated cDNAs (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000310″,”term_id”:”1777425447″,”term_text”:”NM_000310″NM_000310) were inserted into pcDNA3 expression vector (Invitrogen, Life Technologies [LT]) by PCR methods. The sequences of constructs were confirmed by direct sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, LT), on an ABI3130xl automatic DNA Analyzer. SH-SY5Y cells were plated 1 day before transfection at 80% confluence in 35 mm dishes, and then transfected with 250 ng of cDNAs per dish using lipofectamine method (Applied Biosystems, LT) following the manufacturers instructions. Clones which stably overexpressed the different cDNAs were finally isolated through antibiotic selection with 600 g/ml geneticin (G418, Gibco, LT). Cells overexpressing the empty pcDNA3 vector (hereafter also referred as mock) served as a control (Table ?(Table11). Table 1 The compendium of overexpressing SH-SY5Y neuroblastoma cell lines used in the study. cDNAwas assessed by qPCR using inventoried Taqman assay (Hs00165579_m1; Applied Biosystems, LT), and.